Using a rat model of type 2 diabetes (T2D), we showed that at 2 months of T2D the decrease in bone marrow progenitor cell (BMPC) release from diabetic bone marrow (BM) is caused by BM neuropathy and that these changes precede the development of diabetic retinopathy (DR). BM neuropathy was associated with a marked reduction in clock gene expression in the BMPCs themselves which led to diminished repair by these cells and by 4 months of T2D resulted in the hallmark feature of diabetic retinopathy (DR), acellular retinal capillaries. Diabetic BMPC dysfunction was corrected by increasing levels of bioavailable nitric oxide (NO) towards normal non-diabetic levels. Since NO modulates clock gene expression, the reduced levels of bioavailable NO results in altered clock gene expression. Plaminogen activator inhibitor (PAI-1), an important modulator of hematopoetic stem cell maturation and release from the BM is an immediate downstream metabolic regulator controlled by clock genes. PAI-1 demonstrates a robust circadian pattern in health, but this pattern is altered in diabetes and elevated levels are produced by endothelial cells (EC) from diabetics. We believe this NO-modulated clock gene dysfunction leads to the loss of circadian regulation of PAI-1 and represents an underlying mechanism and therapeutic target for DR and atherosclerosis, which are the major causes of blindness and mortality in diabetics, respectively. In this application, we propose the following hypothesis: In diabetes, a reduction in bioavailable NO in ECs and BMPCs causes a diminished amplitude and frequency of the oscillatory pattern of the clock proteins, BMAL1 and PER-2, leading to a loss of circadian regulation of PAI-1 and subsequent further diminution of NO levels in EC and BMPCs. Decreasing NO bioavailability and persistent increase in PAI-1 in the vasa nervorum leads to development of BM neuropathy which results in defective EPC mobilization further exacerbating end organ complications. To test our hypothesis, we propose the following aims: 1) to determine whether NO bioavailability can affect the molecular clock and circadian rhythms in vascular ECs and BMPCs and to determine whether levels of NO will have direct and indirect effects on clock gene expression via S-nitrosylation of essential clock proteins such as BMAL1 and PER-2; 2) to determine whether specific clock gene knock-out of the endothelium and BMPC recapitulates the diabetic vascular phenotype of accelerated injury and reduced repair; and 3) to determine whether dysregulation of NO and PAI-1 within the vasa nervorum leads to BM neuropathy and loss of circadian release of BMPCs into the circulation. PUBLIC HEALTH RELEVANCE: This proposal represents a paradigm shift in our understanding of the pathogenesis of diabetic micro- and macro-vascular complications. We are testing whether circadian oscillators play a pivotal role in endothelial cell and bone marrow progenitor cells homeostasis. We believe that when disrupted in diabetes, they must be restored before vascular health is achieved. We specifically identify plasminogen activator inhibitor as a novel therapeutic target in diabetes to restore vascular function and circadian oscillation of nitric oxide in vascular and bone marrow cells.